Diagnostic agents for pancreatic exocrine function

ABSTRACT

The present invention provides a  13 C-labeled oligosaccharide or polysaccharide or a salt thereof excluding U- 13 C-maltose,  13 C-starch, 1- 13 C-maltotetraose and 1- 13 C-amylose; a derivative of the  13 C-labeled oligosaccharide or polysaccharide or salt thereof; a  13 C-labeled inclusion complex or a salt thereof, which comprises a cyclodextrin or a modified derivative thereof as a host molecule; a  13 C- or  14 C-labeled fluorescein ester compound or a salt thereof; and a diagnostic agents for pancreatic exocrine function comprising these compounds  13 C- or  14 C-labeled.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds useful as diagnosticagents for pancreatic exocrine function and their use.

2. Background of the Invention

“Pancreatic exocrine function tests” are useful for the diagnosis ofpancreatic diseases such as chronic and acute pancreatitis andpancreatic cancer. They are also useful to assess the condition andprognosis of patients and to control the medication: The generaldescriptions are found in Arvanitakis and Cooke, Gastroenterology,74:932 (1978); Niederau and Grendell, Gastroenterology, 88:1973 (1985);Goldberg, Bull. Mol. Biol. Med., 15:1 (1990); Lankisch, Int. J.Pancreatology, 14:9 (1993); Bank and Chow, Gastroenterologist, 2:224(1994); and Steer et al., New Eng. J. Med., 332:1482 (1995).

At present, “Gold standard” of the pancreatic exocrine function testinvolves inserting a tube through the mouth to the duodenum to collectthe duodenal juice. Now, the secretin test is generally utilized whereinsecretin is intravenously administered to stimulate the secretion of thepancreatic juice prior to the collection. This method is highly accuratesince the amount and components of the pancreatic juice are directlyanalyzed. However, this method can not be used repeatedly or used forscreening because of the very strong stress caused on the subjects. Itis available at only a relatively small number of medical centers havinghighly skilled physicians. Further, since this method requiresfluoroscopic tube placement during the collection of the duodenal juice,there is the problem of X ray exposure.

On the other hand, a test for quantifying pancreatic exocrine enzymesfrom the pancreas into the blood is clinically employed for screeningpancreatic diseases (The Merck Manual 16th edition). However, theincrease of the pancreatic exocrine enzymes in the blood is onlyobserved at the initial stage of acute pancreatitis or at therecrudescent stage of chronic pancreatitis and does not always reflectthe ability of the pancreas to secrete pancreatic exocrine enzymes.Further, the increase of pancreatic exocrine enzymes in the blood maysometimes not be detected due to serum turbidity in pancreatitisaccompanied by hyperlipemia.

Accordingly, simple methods which require no insertion of a tube areutilized for repetition and screening tests. One of them is thepancreolauryl test (PLT) wherein a synthetic substrate FDL (fluoresceindiraulate, dilaurylfluorescein) for cholesterol ester hydrolase,esterase, secreted from the pancreas is orally administered and urine isaccumulated for 10 hours followed by measuring the amount of adegradation product fluorescein excreted into the urine: U.S. Pat. No.3,917,812; Barry et al., Lancet (1982) October 2, p. 742; Scharpe andIliano, Clin. Chem., 33:5 (1987). However, this method requires a longtime to carry out the test and therefore can not often be performed onoutpatients and is not suitable in physical examinations.

Under these circumstances, there is a need for the development of asimple method for testing the pancreatic exocrine function which impartslow stress on subjects and gives accurate results soon.

On the one hand, the ¹³C-breath test wherein a ¹³C-labeled starch isadministered has been recently considered to be employed in the test forthe pancreatic exocrine function: Hiele et al., Gastroenterology, 96:503(1989); Dewit et al., Pediatric Res., 32:45 (1992); and Z.Gastroenterol., 35:187 (1997). In the enteric tract, starch is degradedefficiently to glucose by the cleavage at any internal α-1,4 glucosidelinkage with α-amylase secreted from the pancreas and by the action ofenzymes such as α-glucosidase (maltase) of mucosal epithelial cells ofthe small intestine and absorbed: Essentials of Human Metabolism, 2nded., W. C. McMurray, Harper & Row Publishers, NY. The ¹³C-breath testwherein a ¹³C-labeled starch is administered utilizes the phenomenonthat after the ¹³C-labeled starch is degraded in the digestive tract, itis absorbed and decarboxylated by metabolic action in the body togenerate ¹³CO₂ which is excreted into the breath, and it is a safe andsimple method. However, any ¹³C-labeled oligosaccharide orpolysaccharide other than ¹³C-labeled starch has not yet been studied.

Since there is an α-glucosidase (maltase) in mucosal epithelial cells ofthe small intestine, which cleaves a non-reducing terminalα-1,4-glucoside linkage (Enzyme Handbook, Springer-Verlag, Berlin),starch is degraded into glucose sequentially from the non-reducingterminal and absorbed only by the action of enzymes such as theα-glucosidase (maltase), even without the action of α-amylase. Thus,starch is subject to the action of a non-pancreatic-exocrine enzymeα-glucosidase (maltase) of mucosal epithelial cells of the smallintestine and therefore the ¹³C-labeled starch breath test does notreflect the pancreatic exocrine function only. Accordingly, it would bemore preferred if a substrate compound specific for α-amylase in thedigestive tract is selected.

Accordingly, an object of the present invention is to provide adiagnostic agent for pancreatic exocrine function which leads to a testfor the pancreatic exocrine function imparting low stress on subjectsand yields the results in a short period of time.

It is another object of the present invention to provide a diagnosticagent for pancreatic exocrine function which is specific to theα-amylase secretion ability.

It is a further object of the present invention to provide a novelcompound usable in the pancreatic exocrine function test.

SUMMARY OF THE INVENTION

The present inventors have found that the pancreatic exocrine functiontest can be carried out by orally administering a ¹³C-labeledoligosaccharide or a ¹³C-labeled inclusion complex or a ¹³C-labeledfluorescein ester compound to a rat with chronic pancreatitis andmeasuring the ¹³C concentration in the exhaled CO₂ after administration.Thus, the present invention has been completed.

Accordingly, the present invention provides a ¹³C-labeledoligosaccharide or polysaccharide or a salt thereof or a derivativethereof or a C¹³C-labeled inclusion complex other than U-¹³C-maltose,¹³C-starch, 1-¹³C-maltotetraose and 1-¹³C-amylose.

Also, the present invention provides a diagnostic agent for pancreaticexocrine function comprising a ¹³C- or ¹⁴C-labeled oligosaccharide orpolysaccharide or a salt or a derivative thereof, or a ¹³C- or¹⁴C-labeled inclusion complex or a salt thereof other than ¹³C-starch.

Moreover, the present invention provides a diagnostic agent forpancreatic exocrine function comprising a compound which is degradedwith α-amylase but not degraded with α-glucosidase.

Furthermore, the present invention provides a ¹³C- or ¹⁴C-labeldfluorescein ester compound or a salt thereof.

Also, the present invention provides a diagnostic agent for pancreaticexocrine function comprising a ¹³C- or ¹⁴C-labeled fluorescein estercompound or a pharmaceutically acceptable salt thereof.

The subject matters of the present invention are as follows.

(1) A ¹³C-labeled oligosaccharide or polysaccharide or a salt thereofexcluding U-¹³C-maltose, ¹³C-starch, 1-¹³C-maltotetraose and1-¹³C-amylose.

(2) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to (1), which is hydrolyzed with α-amylase.

(3) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to (2), which is not hydrolyzed with α-glucosidase.

(4) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to any one of (1) to (3), wherein at least one sugar moleculeconstituting the oligosaccharide or polysaccharide is ¹³C-labeled.

(5) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to any one of (1) to (3), wherein at least one sugar moleculeconstituting the oligosaccharide or polysaccharide is modified with atleast one ¹³C-labeled modifying group.

(6) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to any one of (1) to (5), which is a linear or branchedoligosaccharide or polysaccharide.

(7) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to any one of (1) to (5), which is a cyclic oligosaccharide orpolysaccharide.

(8) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to (6), which is modified at the non-reducing terminal.

(9) The ¹³C-labeled oligosaccharide or polysaccharide or salt thereofaccording to (1), which is a ¹³C-cyclodextrin orβ-galactosyl-¹³C-maltooligosaccharide.

(10) A derivative of the ¹³C-labeled oligosaccharide or polysaccharideor salt thereof according to any one of (1) to (9).

(11) A ¹³ C-labeled inclusion complex or a salt thereof, which comprisesa cyclodextrin or a modified derivative thereof as a host molecule.

(12) The inclusion complex or salt thereof according to (11), whereinthe host molecule is ¹³C-labeled.

(13) The inclusion complex or salt thereof according to (11), whereinthe guest molecule is ¹³C-labeled.

(14) The inclusion complex or salt thereof according to any one of (11)to (13), wherein the guest molecule is selected from the groupconsisting of oligosaccharides, amino acids, peptides, organic acids,fatty acids, fatty acid glycerides, vitamins, catechins, carotinoids,flavonoids and cholesterol.

(15) The inclusion complex or salt thereof according to (14), whereinthe guest molecule is selected from the group consisting of¹³C-phenylalanine, benzoylphenylalanyl-¹³C-leucine andbenzoylphenylalanyl-¹³C-leucine methyl ester.

(16) A diagnostic agent for pancreatic exocrine function comprising a¹³C- or ¹⁴C-labeled oligosaccharide or polysaccharide or a salt thereofor a derivative thereof other than ¹³C-starch.

(17) The diagnostic agent for pancreatic exocrine function according to(16), wherein the ¹³C- or ⁴C-labeled oligosaccharide or polysaccharideor salt thereof or derivative thereof is hydrolyzed with α-amylase.

(18) The diagnostic agent for pancreatic exocrine function according to(17) wherein the ¹³C- or ¹⁴C-labeled oligosaccharide or polysaccharideor salt thereof or derivative thereof is not hydrolyzed withα-glucosidase.

(19) The diagnostic agent for pancreatic exocrine function according toany one of (16) to (18), wherein the ¹³C- or ¹⁴C-labeled oligosaccharideor polysaccharide is a linear or branched oligosaccharide orpolysaccharide.

(20) The diagnostic agent for pancreatic exocrine function according to(19), wherein the ¹³C- or ¹⁴C-labeled oligosaccharide or polysaccharideis modified at the non-reducing terminal.

(21) The diagnostic agent for pancreatic exocrine function according toany one of (16) to (18), wherein the ¹³C- or ¹⁴C-labeled oligosaccharideor polysaccharide is a cyclic oligosaccharide or polysaccharide.

(22) A diagnostic agent for pancreatic exocrine function comprising a¹³C- or ¹⁴C-labeled inclusion complex or a salt thereof having acyclodextrin or a modified derivative thereof as a host molecule.

(23) The diagnostic agent for pancreatic exocrine function according toany one of (16) to (22), wherein the pancreatic exocrine function to bediagnosed is the ability of the pancreas to secrete α-amylase.

(24) The diagnostic agent for pancreatic exocrine function according toany one of (16) to (22), wherein the pancreatic exocrine function to bediagnosed is the ability of the pancreas to secrete α-amylase and atleast one pancreatic exocrine enzyme other than α-amylase.

(25) A ¹³C- or ¹⁴C-labeled fluorescein ester compound or a salt thereof.

(26) The compound or salt thereof according to (25), which is a compoundor salt thereof resulting from a reaction of a ¹³C- or ¹⁴C-labeled acidwith fluorescein at both or either of the two hydroxyl groups at 3′ and6′ positions to form an ester linkage.

(27) The compound or salt thereof according to (26), wherein the acid isa carboxylic acid.

(28) The compound or salt thereof according to (27), wherein thecarboxylic acid is a fatty acid.

(29) The compound or salt thereof according to (28), wherein the fattyacid has 2 to 16 carbon atoms.

(30) The compound or salt thereof according to (29), wherein the fattyacid is selected from the group consisting of lauric, acetic andoctanoic acids.

(31) The ¹³C-labeled fluorescein ester compound or salt thereofaccording to (25), which is selected from the group consisting of thefollowing compounds:

(a) ¹³C-dilaurylfluorescein;

(b) ¹³C-diacetylfluorescein; and

(c) ¹³C-dioctanoylfluorescein.

(32) A diagnostic agent for pancreatic exocrine function comprising a¹³C- or ¹⁴C-labeled fluorescein ester compound or a pharmaceuticallyacceptable salt thereof.

(33) The diagnostic agent for pancreatic exocrine function according to(32), comprising a compound or salt thereof resulting from a reaction ofa ¹³C- or ¹⁴C-labeled acid with fluorescein at both or either of the twohydroxyl groups at 3′ and 6′ positions to form an ester linkage.

(34) The diagnostic agent for pancreatic exocrine function according to(33), wherein the acid is a carboxylic acid.

(35) The diagnostic agent for pancreatic exocrine function according to(34), wherein the carboxylic acid is a fatty acid.

(36) The diagnostic agent for pancreatic exocrine function according to(35), wherein the fatty acid has 2 to 16 carbon atoms.

(37) The diagnostic agent for pancreatic exocrine function according to(32), which is selected from the group consisting of the followingcompounds:

a) ¹³C-dilaurylfluorescein;

(b) ¹³C-diacetyltluoresceirt; and

(c) ¹³C-dioctanoylfluorescein,

(38) The diagnostic agent for pancreatic exocrine function according toany one of (32) to (37), wherein the ¹³C- or ¹⁴C-labeled fluoresceinester compound or pharmaceutically acceptable salt thereof is subjectedto the action of the pancreatic exocrine cholesterol ester hydrolase andpancreatic exocrine esterase and decarboxylated to generate ¹³CO₂ or¹⁴CO₂.

(39) A ¹³C-labelled oligosaccharide or polysaccharide or salt thereofaccording to any one of (1) to (10) for use in a method of diagnosis.

(40) An inclusion complex according to any of claims (11) to 15 for usein a method of diagnosis.

(41 ) A ¹³C or ¹⁴C-labelled fluorescein ester compound or salt thereofaccording to any one of (25) to (31) for use in a method of diagnosis.

(42) A compound according to any one of (39) to (41) wherein thediagnosis is the diagnosis of pancreatic exocrine function.

The term “oligosaccharide” refers to a sugar having two to ten or moremonosaccharides polmerized. These monosaccharides may be modified.

The term “polysaccharide” refers to a sugar having monosaccharidespolmerized at a degree of polymerization of at least 10. Thesemonosaccharides may be modified.

The term “linear oligosaccharide or polysaccharide” refers to anoligosaccharide or polysaccharide having a linear chain structure suchas maltooligosaccharides (e.g., maltotriose, maltotetraose, etc.) andpolysaccharides (e.g. amylose, etc.).

The term “branched oligosaccharide or polysaccharide” refers to anoligosaccharide or polysaccharide having a branched structure such asamylopectin and glycogen.

The term “cyclic oligosaccharide or polysaccharide” refers to anoligosaccharide or polysaccharide having a cyclic structure such ascyclodextrin.

The term “non-reducing terminal” refers to the terminal at the side atwhich the carbon atom at position 1 of a sugar residue is involved inbinding of the sugar chain.

This specification includes part or all of the contents as disclosed inthe specifications and/or drawings of Japanese Patent Application Nos.10-2712152, 10-271253, 11-261979 and 11-263300 which are prioritydocuments of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a ¹³C-NMR spectrum of ¹³C-cyclodextrin.

FIG. 2 shows the time course of degree of increase of the ¹³Cconcentration in the exhaled CO₂ (Δ¹³C (%)) after administration of¹³C-labeled cyclodextrin. At 0 minute, ¹³C-labeled cyclodextrin wasorally administered (75 mg/kg) to chronic pancreatitis rats (n=2, ) andcontrol rats (n=4, ◯). The bar indicates SD.

FIG. 3 shows a ¹³C-NMR spectrum of galactosyl ¹³C-maltohexaose.

FIG. 4 shows the time course of degree of increase of the ¹³Cconcentration in the exhaled CO₂ (Δ¹³C (%)) after administration of¹³C-galactosyl maltohexaose. At 0 minutes, ¹³C-galactosyl maltohexaosewas orally administered (75 mg/kg) to chronic pancreatitis rats (n=3, )and control rats (n=4, ◯). The bar indicates SD.

FIG. 5 shows the time course of degree of increase of the ¹³Cconcentration in the exhaled CO₂ (Δ¹³C (%)) after administration of¹³C-labeled phenylalanine/hydroxypropyl-β-cyclodextrin(1-¹³C-Phe/hp-β-CD) inclusion complex. At 0 minutes, 1-¹³C-Phe/hp-β-CDinclusion complex was orally administered (59.76 mg/kg of 1-¹³C-Phe) tochronic pancreatitis rats (n=4, ) and control rats (n=4, ◯). The barindicates SD.

FIG. 6 is a powder X-ray diffraction spectrum of benzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrin (Bz-Phe-(¹³C-Leu)/γ-CD) inclusioncomplex.

FIG. 7 shows the time course of degree of increase of the ¹³Cconcentration in the exhaled CO₂ (Δ¹³C (%)) after administration ofbenzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrin(Bz-Phe-(¹³C-Leu)/γ-CD) inclusion complex. At 0 minute,Bz-Phe-(¹³C-Leu)/γ-CD was orally administered (100 mg/kg ofBz-Phe-(¹³C-Leu)) to chronic pancreatitis rats (n=2, ) and control rats(n=2, ◯) The bar indicates SD.

FIG. 8 is a powder X-ray diffraction spectrum ofbenzoylphenylalanyl[l-¹³C]-leucine methyl ester/γ-cyclodextrin(Bz-Phe-(³C-Leu)Me/γ-CD) inclusion complex.

FIG. 9 shows the time course of degree of increase of the ¹³Cconcentration in the exhaled CO₂ (Δ¹³C (%)) after administration ofbenzoylphenylalanyl[1-¹³C]-leucine methyl ester/γ-cyclodextrin(Bz-Phe-(¹³C-Leu)Me/γ-CD) inclusion complex. At 0 minute,Bz-Phe-(¹³C-Leu)Me/γ-CD was orally administered (70 mg/kg ofBz-Phe-(¹³C-Leu)Me) to chronic pancreatitis rats (n=4, ) and controlrats (n=4, ◯). The bar indicates SD.

FIG. 10 shows the time course of degree of increase of the ¹³Cconcentration in the exhaled CO₂ (Δ¹³C (%)) after administration of¹³C-dilaurylfluorescein (¹³C-FDL). At 0 minute, ¹³C-FDL was orallyadministered (160 mg/kg) to chronic pancreatitis rats (, n=3) andcontrol rats (□, n=3). Bars represent SD.

FIG. 11 shows the time course of degree of increase of the ¹³Cconcentration in the exhaled CO₂ (Δ¹³C (%)) after administration of¹³C-dioctanoylfluorescein (¹³C-FDO). At 0 minute, ¹³C-FDO was orallyadministered (200 mg/kg) to chronic pancreatitis rats (, n=4) andcontrol rats (□, n=4). Bars represent SD.

DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention encompasses ¹³C-labeled oligosaccharide orpolysaccharides or salts thereof, or derivatives thereof or ¹³C-labeledinclusion complex other than U-¹³C-maltose, ¹³C-starch,1-¹³C-maltotetraose and 1-¹³C-amylose. The diagnostic agent forpancreatic exocrine function of the present invention comprises a ¹³C-or ¹⁴C-labeled oligosaccharide or polysaccharide or a salt thereof aderivative thereof other than ¹³C-starch, or a ¹³C- or ¹⁴C-labeledinclusion complex or a salt thereof. Preferably, the ¹³C- or ¹⁴C-labeledoligosaccharide or polysaccharides or salts thereof, or derivativesthereof are modified at the non-reducing terminals or have cyclicstructure. These ¹³C- or ¹⁴C-labeled compounds may be pharmaceuticallyacceptable.

The term “¹³C- or ¹⁴C-labeled” herein means that any carbon atom in thecompound is replaced with a ¹³C or ¹⁴C atom, resulting in a higherabundance ratio of ¹³C or ¹⁴C than the naturally occuring abundanceratio irrespective of the preparation method thereof.

The “oligosaccharide or polysaccharides modified at their non-reducingterminals” refers to oligosaccharide or polysaccharides in which thecarbon atom on their non-reducing terminal glucosyl group is modified.Modifying groups in addition to glucopyranose include modifying groupscomprising oligosaccharides such as galactosyl and digalactosyl groups,alkyl groups, alkoxyl groups such as methoxy and benzyloxy groups,carbamoyl group, pyridylamino group, and ethylidene and benzylidenegroups which are attached to two carbon atoms on the non-reducingterminal glucosyl group.

Examples of the oligosaccharide or polysaccharide or salt thereof, orderivative thereof include paranitrophenyl-6-o-benzyl maltopentaoside,4-nitrophenyl maltohexaoside 4,6-ethylidene glucoside, 4,6-benzylideneα-o-4-nitrophenyl-maltopentaoside,o-6-deoxypyridylamino-α-maltopentaoside andparanitrophenyl-4,6-di-o-(N-ethyl)4-carbamoyl maltopentaoside and so on.

These compounds can be prepared by methods described in the followingliterature:

Carbohydrate Research, 176 (1988), 107-115;

Japanese Patent Application Laying Open No. 3-91496;

U.S. Pat. No. 4,649,108;

Journal of Chromatography, 336 (1984), 368-373; and

Japanese Patent Application Laying Open No. 8-291

In the preparation, a ¹³C- or ¹⁴C-labeled maltooligosaccharide (whichmay be commercially available or be prepared by a known method ) can beused as a starting material to give a ¹³C- or ¹⁴C-labeled one.

For example, galactosyl oligosaccharide, which is an oligosaccharidemodified at the non-reducing terminal, may be obtained by adding lactoseto an oligosaccharide to bring lactase into action thereon, andcollecting from the reaction mixture by column chromatography (JapanesePatent Application Laying Open Nos. 4-209277, 8-196289 and 10-316697).In the preparation, a ¹³C- or ¹⁴C-labeled oligosaccharide can be used togive a ¹³C- or ¹⁴C-labeled galactosyl oligosaccharide. When a ¹³C- or¹⁴C-galactosyl labeled lactose is used, an galactosyloligosaccharide inwhich the galactosyl group is ¹³C- or ¹⁴C-labeled is obtained.

“Cyclodextrins” herein means α-1,4 linked cyclic oligosaccharidescomposed of glucopyranose units. The number of glucoses forming the ringis not limited at all and α-, β- and γ-cyclodextrins having 6 to 8glucoses are commercially available. The cyclodextrin may have a branch.The hydroxyl group(s) in cyclodextrin may be modified; cyclodextrinshaving such a modifying group are included in the “modified derivativesof cyclodextrin”. Illustratively, the modification includes alkylationsuch as methylation, hydroxyalkylation such as hydroxypropylation,esterification such as acetylation or succinylation, glucosylation,carboxymethyl etherification, phosphoric esterification, sulfobutyletherification, and carboxymethylation. These modified derivatives aredescribed in Loftsson et al., J. Pharmaceu. Sci., 85:1017 (1996); Stellaet al., Pharmaceutical Res., 14:556 (1997); and “Cyclodextrins”supervised by Fujio Toda, published by Sangyo Tosho.

For example, a ¹³C- or ¹⁴C-labeled cyclodextrin may be obtained bybringing a cyclomaltodextrin glucanotransferase into action on anaturally occurring ¹³C-enriched starch derived from C4 plants such ascorn or a commercially available ¹³C- or ¹⁴C-labeled starch andcollecting from the reaction mixture by column chromatography.

Furthermore, a precipitation of ¹³C- or ¹⁴C-labeled β- andγ-cyclodextrin can be obtained by adding bromobenzene to a mixedsolution of ¹³C- or ¹⁴C-labeled cyclodextrin, stirring the mixture at10° C. overnight and subjecting it to centrifugation. This precipitationis washed with bromobenzene-saturated ice water, concentrated by vaporevaporation and left to stand, resulting in ¹³C- or ¹⁴C-labeledβ-cyclodextrin. The mother liquor is treated with glucoamylase and mixedwith cyclohexane. The supernatant is concentrated and mixed withn-propanol to give ¹³C- or ¹⁴C-labeled cyclodextrin.

A modified derivative of a cyclodextrin may be prepared in the followingmanner. For example, potassium hydroxide is added to an aqueous solutionof β-cyclodextrin to make it alkaline and the solution is heated to 70to 80° C. 2-Chloroethanol or 3-chloropropanol is then added. The mixtureis then cooled to room temperature and neutralized and active carbon isadded and filtered. The filtrate is concentrated and dried. DMF is addedto the residue and insolubles are removed. Addition of acetone yieldshydroxyethyl- or hydroxypropylcyclodextrin: Irie et al., PharmaceuticalRes., 5:713 (1988). In the preparation, a ¹³C- or ¹⁴C-labeledcyclodextrin can be used to give ¹³C- or ¹⁴C-labeled hydroxyethylcyclodextrin or hydroxypropyl cyclodextrin.

The “cyclodextrin inclusion complex” refers to a cyclodextrin or amodified derivative thereof having any compound included in the cavityin the cyclic molecule. The compound included may be amino acids, fattyacids, organic acids, catechins, vitamins, carotenoids, flavonoids,cholesterols, and modified derivatives thereof. Additional examples ofthe compound included are oligosaccharides, peptides, fatty acidglycerides and modified derivatives thereof.

The ¹³C- or ¹⁴C-labeled cyclodextrin inclusion complex may be one inwhich any carbon atom in the included compound is replaced with a ¹³C or¹⁴C atom, resulting in a higher abundance ratio of ¹³C or ¹⁴C in thecyclodextrin inclusion complex than the naturally occuring abundanceratio. However, the cyxlodextrin or modified derivative thereof per semay be ¹³C- or ¹⁴C-labeled. Methods for the preparation thereof are notlimited.

Examples of cyclodextrin inclusion complexes reported heretofore includethe following (host molecules are not limited to ones indicated in thecomplexes):

Phenylalanine/β-cyclodextrin inclusion complex

Tryptophanla/α-cyclodextrin inclusion complex

Histidine/α-cyclodextrin inclusion complex

Cinnalidine/β-cyclodextrin inclusion complex

Ferulic acid/β-cyclodextrin inclusion complex

Phenylalanyl leucine/β-cyclodextrin inclusion complex.

Other guest molecules include phenol, hydroxy benzoic acid,benzaldehyde, methyl sulfoxide, benzoic acid, aniline, amino benzoicacid, methyl benzoic acid, nitrophenol, pyridine, acetic acid, alcohols(e.g., ethanol, propanol, butanediol, etc.), prostandin, benexatehydrocholoride, nitroglycerin, limaprost and the like, which aredescribed in “Cyclodextrin” edited by Fujio Toda, Sangyo Tosho. Thecyclodextrin inclusion complex can be obtained by mixing purified waterwith a mixture of a host compound and a guest compound at a ratio equalto that of the host to the guest in the inclusion complex (e.g., at anequivalent molar ratio in the case where the ratio of the host to theguest is 1:1), stirring the resulting mixture for about 12 hours, andsubjecting it to a spray dry treatment.

The ¹³C- or ¹⁴C-labeled oligosaccharide or polysaccharide orcyclodextrin inclusion complex may be also obtained in the form of asalt. Such salts include those with inorganic acids such ashydrochloric, sulfuric and phosphoric acids; those with organic acidssuch as formic, acetic, propionic, glycolic, succinic, malic, tartaric,citric and trifluoroacetic acids; those with alkali metals such assodium and potassium; those with alkaline earth metals such as calcium;and those with ammonium or organic amines such as ethanolamine,triethylamine and dicyclohexylamine.

The present invention also encompasses ¹³C- or ¹⁴C-labeled fluoresceinester compounds or salts thereof.

Fluorescein (CA Name: 3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′-[9H]xanthen-3-one) isrepresented by the following structural formula:

The “fluorescein ester compound” refers to a compound resulting from areaction of an acid with fluorescein at the hydroxyl group(s) to form anester linkage. Fluorescein and the acid may be modified.

The “¹³C- or ¹⁴C-labeled” means that at least one carbon atom in thefluorescein ester compound is replaced with a ¹³C- or ¹⁴C atom,resulting in a higher abundance ratio of the ¹³C- or ¹⁴C atom than thenaturally occuring abundance ratio.

In one embodiment of the present invention, the ¹³C- or ¹⁴C-labeledfluorescein ester compound or salt thereof is a compound resulting froma reaction of a ¹³C- or ¹⁴C-labeled acid with fluorescein or a saltthereof at both or either of the hydroxyl groups at 3′ and 6′ positionsto form an ester linkage.

An example of the ¹³C- or ¹⁴C-labeled acid may be a carboxylic acid,preferably a fatty acid. The “fatty acid” herein refers to a compoundrepresented by the formula R—COOH in which R is an aliphatic group whichmay optionally have a branch(es) and/or a double bond(s). The number ofcarbons in the fatty acid is preferably 2 to 16.

Examples of the fatty acid include acetic, octanoic and lauric acids butare not limited thereto.

Examples of the ¹³C- or ¹⁴C-labeled fluorescein ester compound include¹³C-dilaurylfluorescein, ¹⁴C-diacetylfluorescein,¹³C-dioctanoylfluorescein, and the like.

The ¹³C- or ¹⁴C-labeled fluorescein ester compound may be prepared inthe following manner.

For example, fluorescein is dissolved in chloroform and an equal ortwice molar amounts of a ¹³C- or ¹⁴C-labeled fatty acid chloride isadded. Then, a chloroform solution containing pyridine is dropwise addedand stirred and heated under dark. After the reaction, the solvent isdistilled out. Column chromatography and recrystallization yield the¹³C- or ¹⁴C-labeled fluorescein ester compound.

The ¹³C- or ¹⁴C-labeled fluorescein ester compound may be prepared inthe form of a salt. The salts may include sodium and potassium salts.

The diagnostic agent for pancreatic exocrine function according to thepresent invention may be formulated from the ¹³C- or ¹⁴C-labeledcompound alone or in combination with an excipient or carrier into anoral preparation such as a tablet, capsule, powder, granule or liquid.The excipient or carrier may be any pharmaceutically acceptable oneordinarily used in this field and its nature and composition may beappropriately chosen. For example, water may be used as a liquidcarrier. Solid carriers include cellulose derivatives such ashydroxypropyl cellulose, and organic acid salts such as magnesiumstearate. Also, freeze-dried preparations may be used.

The ¹³C- or ¹⁴C-labeled compound is contained in the preparation invariable amounts depending on the nature of the preparation, butgenerally in an amount of 1 to 100% by weight, preferably 50 to 100% byweight. In a capsule, tablet, granule or powder preparation, the ¹³C- or¹⁴C-labeled compound is contained in the preparation in an amount ofabout 10 to 100% by weight, preferably 50 to 100% by weight, the balancebeing a carrier.

The dose of the diagnostic agent for pancreatic exocrine functionaccording to the present invention should be sufficient to confirm anincrease of ¹³CO₂ or ¹⁴CO₂ in the breath after the administration. Itwill vary depending upon the age and body weight of a subject and thepurpose of the test. For example, the unit dose may be 1 to 2000 mg/kgof body weight for an adult.

The test using the agent for pancreatic exocrine function according tothe present invention is carried out with administering to a subject the13C- or ¹⁴C-labeled compound. A test is possible, in which theconcentration of a ¹³C- or ¹⁴C-labeled compound is measured in serum,urine or stool after the administration, however, a breath test isdesirable in which an increase in ¹³C or ¹⁴C concentration is measuredin the exhaled CO₂ after the administration. When the ¹³C- or¹⁴C-labeled compound is administered to a subject, a test meal or thelike may be pre-ingested by the subject to induce secretion ofpancreatic enzymes. The ¹³C- or ¹⁴C-labeled compound may be administeredtogether with the test meal or the like. Also, a plurality of the ¹³C-or ¹⁴C-labeled compound may be combined for use. Concretely, in the caseof ¹³C, the ¹³C concentration is determined in the exhaled CO₂ after theadministration, then the pancreatic exocrine function is diagnosed fromeither the data of the degree of increase (Δ¹³C (%)) of the ¹³Cconcentration in the exhaled CO₂ at predetermined times (e.g., 5, 10 and15 minutes) after the administration, or the data integrated orassociated with the time course (onset slope, change in slope, peaktime, etc.) in the degree of increase (Δ¹³C (%)) of the ¹³Cconcentration in the exhaled CO₂ during a predetermined period after theadministration. In the case of ¹⁴C, the ¹⁴C concentration, i.e.,radioactivity, is determined in the exhaled CO₂ after theadministration; and the pancreatic exocrine function is diagnosed fromeither the data of the quantity of radioactivity in the exhaled CO₂ atpredetermined times (e.g., 5, 10 and 15 minutes) after theadministration, or the data integrated or associated with the timecourse (onset slope, change in slope, peak time, etc.) in the rateincrease of radioactivity in the exhaled CO₂ during a predeterminedperiod after the administration.

These test methods utilize the phenomenon that when the ¹³C- or¹⁴C-labeled compound is administered to a subject, the compound isabsorbed through the digestive tract after it is degraded by the actionof the pancreatic exocrine α-amylase and/or esterase, and decarboxylatedby metabolic action in the body to generate ¹³CO₂ or ¹⁴CO₂ which isexcreted into the breath.

When a cyclodextrin inclusion complex, in which an oligosaccharide,peptide, fatty acid glyceride or a modified derivative thereof isincluded, is used, the first reaction of the degradation is the cleavageof the cyclodextrin by α-amylase and the oligosaccharide, peptide, fattyacid glyceride or modified derivative thereof released in associationwith the cleavage is then degraded by the action of the pancreaticexocrine α-amylase, protease, lipase or the like to be absorbed throughthe digestive tract and decarboxylated by metabolic action in the bodyto generate ¹³CO₂ or ¹⁴CO₂ which is excreted into the breath.

The ¹³C concentration in the exhaled CO₂ can be determined by gaschromatography-mass spectrometry (GC-MS), infrared spectroscopy, massspectrometry, photoelectric acoustic spectroscopy, NMR (nuclear magneticresonance), and other methods.

The ¹⁴C concentration or radioactivity in the exhaled CO₂ may bemeasured from the breath of a subject, directly or after trapping CO₂ ina solvent, with a GM counter, a liquid scintillation counter, a solidscintillation counter, autoradiography, an ionization chamber, or thelike.

The diagnostic agent for pancreatic exocrine function according to thepresent invention is particularly effective in the diagnosis of theα-amylase and/or esterase secretion ability of the pancreas.

Hereinbelow, the present invention is illustrated in more detail by thefollowing examples; however, the scope of the present invention shallnot be limited by the example.

EXAMPLES Example 1 Preparation of ¹³C-labeled Cyclodextrin

¹³C-labeled starch (Chlorella Industry, Algal Starch (water-soluble),Lot No. 8031,S, U-¹³C:98.6 atom %, Starch Content: 93.5%) was dissolvedin 50 mM acetate buffer (pH 5.4) at a concentration of 5% (w/v) and 100Units of cyclomaltodextrin glucanotransferase (Hayashibara) was addedthereto and reacted at 40° C. for 24 hours. After the enzyme wasinactivated with the treatment at 100° C. for 15 minutes, glucoamylasewas added and reacted at 40° C. for 1 hour to decompose components otherthan ¹³C-labeled cyclodextrin into glucose. After the reaction wascompleted, the reaction mixture was treated at 100° C. for 15 minutes toinactivate the glucoamylase.

The solution was applied to a carbon column (2.5 cm×25 cm) and thecolumn was washed with 500 ml of water. A 15% ethanol solution and a 40%ethanol solution were sequentially applied to recover the ¹³C-labeledcyclodextrin in the 40% ethanol solution eluted fractions. The resulting¹³C-labeled cyclodextrin solution was distilled to remove the solvent,dissolved in a small amount of water and freeze-dried.

The product was mixed with para-nitrophenol to confirm to becyclodextrin from an increase in absorbance at a wave length of 450 nm.

The ¹³C-labeled position was confirmed by ¹³C-NMR (FIG. 1).

¹³C-NMR (DMSO-d6, 300 MHz)

38.5-40.2 ppm Dimethyl sulfoxide

59.5-60.1 ppm Position 6 of glucose residue

71.4-73.1 ppm Positions 3, 4 and 5 of glucose residue

80.9-82.5 ppm Position 2 of glucose residue

101.5-102.1 ppm Position 1 of glucose residue

HPLC analysis (Shodex Asahipak GS-220 HQ) of the resulting ¹³C-labeledcyclodextrin revealed that it was a mixture of 45% of α-cyclodextrin,45% of β-cyclodextrin and 10% of γ-cyclodextrin.

Example 2 ¹³C-labeled Cyclodextrin Breath Test

¹³C-labeled cyclodextrin breath test was carried out wherein ¹³C-labeledcyclodextrin prepared in Example 1 was orally administered to chronicpancreatitis and control rats and the time course of the ¹³Cconcentration in the exhaled CO₂ after the administration was measured.

According to Mundlos et al. (Mundlos et al., Pancreas, 1:29 (1986)), thechronic pancreatitis rats were prepared by injecting oleic acid into thepancreatic duct of Wistar male rats of 5 weeks old and kept for 3 weeks.Rats in which midline incision was made on the abdomen were used as thecontrol.

The chronic pancreatitis and control rats of 8 weeks old, which fastedovernight, were fixed without anesthesia in a rat holder for a microwaveirradiation apparatus. The breath was collected at a rate of about 100to 300 ml/min using a stroke pump (Variable Stroke Pump VS-500, ShibataKagaku Kogyo) and introduced directly to a flow cell of a ¹³CO₂ analyzerEX-130S (Nihon Bunko) to measure ¹³C atom % and the carbon dioxide gasconcentration continuously. A Perma Pure drier (MD-050-12P, Perma PureInc.) was set between the rat holder and the stroke pump to remove outwater vapor in the breath. After the CO₂ concentration was stabilized,the rat was once removed out of the rat holder and an aqueous solutionof ¹³C-labeled cyclodextrin was administered (75 mg/kg, 5 ml/kg) intothe stomach using an oral sonde.

Δ¹³C (%) was calculated from the ¹³C concentration in the exhaled Co₂ ateach time point (¹³C tmin) and the ¹³C concentration in standard CO₂(¹³C std) according to the following equation:

Δ¹³C (%)=[(¹³C tmin− ¹³C 0 min)/ ¹³C std]×1000

In both the control and chronic pancreatitis rats, the Δ¹³C (%) valuescontinued to increase for 30 minutes. However, the increase in Δ¹³C (%)value of the chronic pancreatitis rats was smaller than the control rats(FIG. 2). At 15 minutes, the Δ¹³C (%) value of 12.88±4.25 in the chronicpancreatitis rats was significantly smaller than the value of 30.39±5.29in the control rats (p<0.05, ANOVA with Fischer LSD).

Example 3 Preparation of ¹³C-labeled Galactosylmaltohexaose

¹³C-labeled starch (Chlorella Industry, 4.65 g) was dissolved in a 50 mMacetate buffer (pH 5.4) at a concentration of 0.5% (w/v) and 186 Unitsof cyclomaltodextrin glucanotransferase (Hayashibara) was added theretoand reacted at 40° C. for 2 hours and 20 minutes. The enzyme wasinactivated with the treatment at 95° C. for 15 minutes and the productwas purified by Sephadex G-25. This procedure was repeated 4 times toyield 4.39 g of ¹³C-α-cyclodextrin (yield 23.6%).

Pyridine (200 mL) was added to 4.39 g of the resulting¹³C-α-cyclodextrin and ice cooled. Acetic anhydride (100 mL) was addedthereto. After 30 minutes, the reaction mixture was removed out of theice bath and then stirred at room temperature for 36 hours. To theresidue obtained by concentration under reduced pressure, toluene wasadded and azeotropically distilled. This procedure was repeated threetimes. Ethyl acetate and water were added to the residue to extract. Theorganic layers were combined, dried over anhydrous magnesium sulfate andfiltered and the filtrate was concentrated under reduced pressure. Theresulting residue was purified by silica gel column chromatography toyield 5.1 g of peracetylated ¹³C-α-cyclodextrin.

The peracetylated ³C-α-cyclodextrin (3.25 g) was dissolved in aceticanhydride (49.8 mL) and sulfuric acid (1.02 mL) and stirred underheating at 55° C. After 5 hours, the reaction mixture was ice cooled andpyridine (5.1 mL) was added. The reaction mixture was concentrated underreduced pressure and toluene was added to the resulting residue. Thisprocedure was repeated three times. Chloroform and water were added tothe residue to extract. The organic layers were combined, dried overanhydrous magnesium sulfate and filtered and the filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedby silica gel column chromatography. This procedure was repeated twiceand the collected starting material was again used in the ring-openingreaction to yield 4.80 g of peracetylated ¹³C-maltohexaose from 6.50 gin total of the starting material.

The peracetylated ¹³C-maltohexaose (3.76 g) was dissolved in 1500 mL ofdry methanol and ice cooled. A solution of 5.18 M sodium methoxide inmethanol (776 μl) was added thereto. After 30 minutes, the reactionmixture was returned to room temperature and stirred. After 20 hours, asolution of 5.18 M sodium methoxide in methanol (388 μl) was added.After 3.5 hours, Amberlyst 15 (16 g) was added to neutralize andfiltered. The resin was washed with methanol and water and the filtrateand washings were combined and concentrated under reduced pressure. Theresulting residue was purified by HPLC (TSK-Gel Amide-80 column) toyield 1.78 g of ¹³C-maltohexaose. The same procedure was repeated twiceto yield 2.03 g of ¹³C-maltohexaose in total.

To 2.03 g of ¹³C-maltohexaose and 710 mg of lactose monohydrate, 20 mMpotassium phosphate buffer (pH 7.0, 6.50 mL) was added and dissolved at40° C. A solution of Biolacta (lactase)(Daiwa Kasei, 0.87 mg) in 20 mMpotassium phosphate buffer (20 μl) was added and allowed to stand at 40°C. After 9.5 hours, the reaction mixture was heated at 95° C. for 15minutes. The enzymic reaction solution was purified by HPLC (TSK-GelAmide-80 column) to yield 239 mg of galactosyl ¹³C-maltohexaose. HPLCanalysis (TSK-Gel Amide-80 column) of the pyridylaminated galactosyl¹³C-maltohexaose revealed that the resulting galactosyl ¹³C-maltohexaosecomprised β-1,4-galactosyl ¹³C-maltohexaose as a main component and notmore than 9% of β-1,6-galactosyl ¹³C-maltohexaose.

The structure was confirmed by ¹³C-NMR (FIG. 3) and mass spectrometry.

¹³C-NMR (D₂O, 270 MHz)

60.3-61.6 ppm Position 6 of 6 glucose residues

67.4 ppm 1,4-Dioxane

70.7-79.6 ppm Positions 2, 3, 4 and 5 of 6 glucose residues

92.4-96.9 ppm Position 1 of the reducing terminal glucose

100.1-101.5 ppm Position 1 of 5 glucose residues

Mass spectrometry (ESI-MS) m/z: 1211.3 (M⁺+Na)

Example 4 Galactosyl ¹³C-labeled Maltohexaose Breath Test

As in Example 2, a galactosyl ¹³C-labeled maltohexaose breath test wascarried out wherein galactosyl ¹³C- labeled maltohexaose obtained inExample 3 was orally administered (75 mg/kg) to chronic pancreatitis andcontrol rats and the time course of the ¹³C concentration in the exhaledCO₂ after the administration was measured.

In both the control and chronic pancreatitis rats, the Δ¹³C (%) valuescontinued to increase for 30 minutes. However, the increase in Δ¹³C (%)value of the chronic pancreatitis rats was smaller than the control rats(FIG. 4). At 10 minutes after the administration, the Δ¹³C (%) value(18.89±17.01%) of in the chronic pancreatitis rats was significantlysmaller than the value (95.57±42.40%) of in the control rats (p<0.05,ANOVA with Fischer LSD).

Example 5 Preparation of [1-¹³C]-phenylalanine/hydroxypropyl-β-cyclodextrin Inclusion Complex

To 5 ml of distilled water, 299 mg of [1-¹³C]-phenylalanine and 1818 mghydroxypropyl-β-cyclodextrin (in a molar ratio of 1:1) were added andheated to 85° C. to make a solution. Thus, a solution of[1-¹³C]-phenylalanine/hydroxypropylβ-cyclodextrin inclusion complex wasprepared wherein when this solution was allowed to cool to 25° C., noprecipitation of phenylalanine was observed while the solubility ofphenylalanine is 148 mg/5 ml at 25° C. On the other hand, if 299 mg of[1-¹³C]-phenylalanine only was added to 5 ml of distilled water andheated to 85° C. to dissolve, phenylalanine was precipitated when thissolution was allowed to cool to 25° C. The increase of solubility ofphenylalanine in the presence of hydroxypropyl-β-cyclodextrin confirmedthe formation of [1-¹³C]-phenylalanine/hydroxypropyl-β-cyclodextrininclusion complex.

Example 6 [1-¹³C]-phenylalanine/hydroxypropyl-β-cyclodextrin InclusionComplex Breath Test

As in Example 2, a [1-¹³C]-phenylalanine/hydroxypropyl-β-cyclodextrininclusion complex breath test was carried out wherein[1-¹³C]-phenylalanine/hydroxypropyl-β-cyclodextrin inclusion complexobtained in Example 5 was orally administered (59.76 mg/kg of[1-¹³C]-phenylalanine) to chronic pancreatitis and control rats and thetime course of the ¹³C concentration in the exhaled CO₂ after theadministration was measured.

The chronic pancreatitis rats were WBN/kob male rats (Japan SLC, Inc.)of 19 weeks old. The control rats were Wistar male rats of 19 weeks old.

The Δ¹³C (%) values of the chronic pancreatitis rats were smaller thanthe control rats during 30 minutes (FIG. 5). At 5 minutes after theadministration, the Δ¹³C (%) value of 38.18±17.46 in the chronicpancreatitis rats was significantly smaller than the value of132.60±26.79 in the control rats (p<0.005, ANOVA with Fischer LSD).

Example 7 Preparation ofBenzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrin Inclusion Complex

After 1 g of 1-¹³C-L-leucine (Masstrace) was dissolved in hydrogenchloride/methanol and refluxed, the resulting ¹³C-L-leucine methyl esterwas suspended in 50 ml of dichloromethane and 1.08 ml of triethylaminewas added dropwise under while being ice-cooled and stirred. Further,2.0 g of N-benzoyl-DL-phenylalanine, 2.34 g of HOBt(1-hydroxy-1H-benzotriazole.H₂O) and 50 ml of dichloromethane wereadded. Then, a solution of 1.49 g of WSC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.HCl) dissolved in 100 mlof dichloromethane was added and stirred for 1 hour under while beingice-cooled and then overnight at room temperature. The completion of thereaction was confirmed by silica gel thin layer chromatography usingchloroform:methanol (95:5) as a developing solvent. The reaction mixturewas concentrated, extracted with ethyl acetate, washed with 1N-HCl, 5%NaHCO₃, and water, dried over magnesium sulfate, and concentrated todryness to yield 2.32 g of benzoylphenylalanyl-[1-¹³C]-leucine methylester.

After 2.32 g of benzoylphenylalanyl-[1-¹³C]-leucine methyl ester wasdissolved in 100 ml of methanol, 6.4 ml of 1N NaOH was added dropwiseunder while being ice-cooled and stirred followed by heating andstirring at 70° C. for 2.5 hours. The completion of the reaction wasconfirmed by silica gel thin layer chromatography usingchloroform:methanol (95:5) as a developing solvent. After the reactionwas completed, the reaction mixture was neutralized with 1N-HCl,concentrated and dissolved in 5% NaHCO₃. After washing with ethylacetate, 5% NaHCO₃ was acidified with 1N-HCl. The reaction mixture wasextracted with ethyl acetate, washed with water, dried over magnesiumsulfate, and concentrated to dryness to yield 1.93 g ofbenzoylphenylalanyl-[1-¹³C]-leucine, which was then recrystallized withethyl acetate.

The structure and ¹³C-labeled position were confirmed by ¹³C-NMR andmass spectrometry.

¹³C-NMR (methanol-d4, 300 MHz): 175.8 ppm (¹³COOH)

Mass spectrometry (m/z): 383 (M⁺), 365, 224, 131, 105, 77 LC-MS (m/z):384 (M⁺+H), 252, 224, 105.

Benzoylphenylalanyl[1¹³C]-leucine was prepared so that the weight ratiothereof to γ-cyclodextrin was 1:4 (1:1 in molar ratio). γ-Cyclodextrinand benzoylphenylalanyl[1-¹³C]-leucine were dissolved in a small amountof purified water and ethanol, respectively, and the both solutions weremixed together, stirred with a stirrer (500 rpm) for 12 hours, and thenspray dried using Pulvis minispray GA-31 (Yamato Kagaku) under theconditions: an inlet temperature of 100° C., an outlet temperature of50° C., a dry air flow rate of 0.45 m³/min, a spray pressure of 1.5kg/cm², and a flow rate of 5 ml/min. The resulting sample was furtherdried under reduced pressure for 24 hours and passed through No. 18sieve (850 μm). Thus, benzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrininclusion complex was obtained as a powder remaining on No. 50 sieve(300 μm).

The structure was confirmed by powder X-ray diffraction using GeigerflexModel 2013 diffractometer (Rigaku Denki) under the conditions: Nifilter, Cu-K α line (30 kV, 20 mA), a scanning speed of 1°/min. In thebenzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrin inclusion complex, nodiffraction peak inherent to benzoylphenylalanyl[1-¹³C]-leucine orγ-cyclodextrin was observed but a halo curve was observed (FIG. 6).

Example 8 Benzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrin InclusionComplex Breath Test

As in Example 2, a benzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrininclusion complex breath test was carried out whereinbenzoylphenylalanyl[1-¹³C]-leucine/γ-cyclodextrin inclusion complexobtained in Example 7 was orally administered (suspended in 0.5% sodiumcarboxymethyl cellulose (CMC-Na) solution, 100 mg/kg ofbenzoylphenylalanyl[1-¹³C]-leucine) to chronic pancreatitis and controlrats and the time course of the ¹³C concentration in the exhaled CO₂after the administration was measured.

The Δ¹³C (%) values of the chronic pancreatitis rats were smaller thanthe control rats during 40 minutes (FIG. 7). At 10 minutes after theadministration, the A ¹³C (%) value of 0.03±1.73 in the chronicpancreatitis rats was smaller than the value of 7.43±4.42 in the controlrats.

Example 9 Preparation of Benzoylphenylalanyl[1-¹³C]-leucine methylester/γ-cyclodextrin inclusion complex

Benzoylphenylalanyl[1-¹³C]-leucine methyl ester (prepared in Example 7)was prepared so that the weight ratio thereof to γ-cyclodextrin was 1:4(1:1 in molar ratio). γ-Cyclodextrin andbenzoylphenylalanyl[1-¹³C]-leucine methyl ester were dissolved in asmall amount of purified water and ethanol, respectively, and the bothsolutions were mixed together, and stirred with a stirrer (500 rpm) for12 hours. Then, the procedures of Example 7 were repeated to yieldbenzoylphenylalanyl[1-¹³C]-leucine methyl ester/γ-cyclodextrin inclusioncomplex.

The structure was confirmed in the same manner as in Example 7. In thebenzoylphenylalanyl[1-¹³C]-leucine methyl ester/γ-cyclodextrin inclusioncomplex, no diffraction peak inherent tobenzoylphenylalanyl[1-¹³C]-leucine methyl ester or γ-cyclodextrin wasobserved but a halo curve was observed (FIG. 8).

Example 10 Benzoylphenylalanyl[1-¹³C]-leucine methylester/γ-cyclodextrin Inclusion Complex Breath Test

As in Example 2, a benzoylphenylalanyl[1-¹³C]-leucine methylester/γ-cyclodextrin inclusion complex breath test was carried outwherein benzoylphenylalanyl[1-¹³C]-leucine methyl ester/γ-cyclodextrininclusion complex obtained in Example 9 was orally administered(suspended in 0.5% CMC-Na solution, 70 mg/kg ofbenzoylphenylalanyl[1-¹³C]-leucine methyl ester, 5 ml/kg) to chronicpancreatitis and control rats and the time course of the ¹³Cconcentration in the exhaled CO₂ after the administration was measured.

The Δ³C (%) values of the chronic pancreatitis rats were smaller thanthe control rats during 40 minutes (FIG. 9). At 10 minutes after theadministration, the Δ¹³C (%) value of 2.24±1.32 in the chronicpancreatitis rats was significantly smaller than the value of 6.73±2.06in the control rats (p<0.05, ANOVA with Fischer LSD).

Example 11 Preparation of ¹³C-dilaurylfluorescein (¹³C-FDL)

Five grams (5 g) of 1-¹³C-lauric acid (Masstrace) was dissolved in drychloroform and 20 fold molar amount of thionyl chloride was addedthereto. This solution was refluxed under heating for 2 hours andchloroform was removed out by an evaporator. Further, thionyl chloridewas distilled out under reduced pressure and the residue was immediatelyused in the subsequent reaction. Fluorescein 2Na in a ⅙ molar amount ofthe lauryl chloride was dissolved in 20 ml of dry acetone and an equalmolar amount of pyridine was added and heated at 45° C. The acidchloride obtained above was dropwise added through a dropping funnelover 30 minutes, during which light was shut out and the temperature waskept at 45° C. After the dropping, the mixture was reacted for 2 hours.

After the reaction, acetone was distilled out and chloroform was thenadded to dissolve the residue. Materials insoluble in chloroform wereremoved out by filtration and the chloroform phase was washedsequentially with water, alkali, water, acid, and water and dried oversodium sulfate. Chloroform was distilled out and the residue waspurified by silica gel column (3 cm×60 cm, chloroform/ether) and activecarbon. The resulting compound was washed with cold methanol to yield¹³C-FDL.

The structure and ¹³C-labeled position were confirmed by ¹³C-NMR andmass spectrometry.

¹³C-NMR (heavy chloroform, 300 MHz): 172.2 ppm (¹³ COOR)

Mass spectrometry (EI-MS) m/z: 698 (M⁺), 288, 287, 271

LC-MS (APCI) m/z: 699 (M⁺+H), 516, 333

Example 12 ¹³C-FDL Breath Test

12-1 Method

A ¹³C-FDL breath test was carried out wherein ¹³C-FDL was orallyadministered to chronic pancreatitis and control rats and the timecourse of the ¹³C concentration in the exhaled CO₂ after theadministration was measured.

According to Mundlos et al. (Mundlos et al., Pancreas, 1:29 (1986)), thechronic pancreatitis rats were prepared by injecting oleic acid into thepancreatic duct of Wistar male rats of 5 weeks old and kept for 3 weeks.Rats in which midline incision was made on the abdomen were used as thecontrol.

The chronic pancreatitis and control rats of 8 weeks old, which fastedovernight, were fixed without anesthesia in a rat holder for a microwaveirradiation apparatus. The breath was collected at a rate of about 100to 300 ml/min using a CO₂ meter (CAPSTER-100) to monitor the CO₂concentration. After the CO₂ concentration was stabilized, the rat wasonce removed out of the rat holder and the ¹³C-FDL dissolved in oliveoil was administered (160 mg/kg, 4 ml/kg) into the stomach using an oralsonde.

The breath was taken out as a sample immediately before theadministration and at every hour for 5 hours after the administrationand the ¹³C concentration in the exhaled CO₂ was analyzed by a gaschromatography-mass spectrometer (GC-MS). The analytic conditions forGC-MS are as follows. The CO₂ concentration in the collected breath washeld at 3±0.5%. GC-MS conditions:

Apparatus: Shimadzu GC-MS QP-5000 (Shimadzu Corporation)

Column: 0.32 mm×25 m (ID×L) fused silica capillary column PORAPLOT Q(CHROMPACK)

Ionization: EI (electron impact)

Vaporization chamber temperature: 60° C.

Column temperature: 60° C.

GC interphase temperature: 230° C.

Carrier gas: He

Carrier gas pressure: 20 KPa

Measurement mode: SIM (selected ion monitoring)

Measured ion: m/z=45, 46, 47

Amount of sample injected: 25 μl

Method for Calculation of ¹³C Concentration

Assuming that the isotopic abundance of oxygen is the same as that whichis naturally occurring, ¹³C concentration was calculated from the peakareas of the ions m/z=45 and 46 according to the following equation:

¹³C concentration (%)=[0.004176−0.0007462a)/(0.9944396 +0.0034298a)]×100

wherein a is an area ratio (A45/A46) of m/z being 45 and 46 (seeJapanese Patent Application Laying Open No.7-120434).

Δ¹³ C (%) was calculated from the ¹³C concentration in the exhaled CO₂at each time point (¹³C tmin) and the ¹³C concentration in standard CO₂(¹³C std) according to the following equation:

Δ¹³C (%)=[(¹³C tmin− ¹³C 0min)/ ¹³C std]×1000

12.2 Results

In both the control and chronic pancreatitis rats, the AΔ¹³C (%) valuecontinued to increase for 5 hours. However, the Δ¹³C (%) values of thechronic pancreatitis rats were smaller than the control rats at eachtime point for 5 hours (FIG. 10). At 3 hours after the administration,the Δ¹³C (%) value of 42.0 ±21.3 in the chronic pancreatitis rats wasvery significantly smaller than the value of 153.3±22.8 in the controlrats (p<0.01). At 5 hours after the administration, the Δ¹³C (%) valueof 95.4±47.1 in the chronic pancreatitis rats was significantly smallerthan the value of 236.3±12.4 in the control rats (p<0.05).

Example 13 Preparation of ¹³C-dioctanoylfluorescein (¹³C¹³C-FDO)

Two grams (2 g) of 1-¹³C-octanoic acid (Masstrace) and 1.52 g offluorescein 2Na were dissolved in dimethylformamide (DMF) and 9.13 g ofbenzotriazol-1-yl-oxy-tris(dimethylamino) phosphoniumhexafluorophosphate (BOP) and 7.2 ml of diisopropylethylamine (DIEA)were sequentially added and stirred at room temperature for 12 hours.After the completion of the reaction was confirmed by TLC, the reactionmixture was extracted with ethyl acetate. The organic layer was dried,concentrated, purified through a column (solvent: 20% ethylacetate/hexane) under dark, and concentrated to yield 1.05 g of ¹³¹³C-FDO.

The structure and ¹³C-labeled position were confirmed by ¹³C-NMR andmass spectrometry.

¹³C-NMR (heavy chloroform, 400 MHz): 171.9 ppm (¹³COOR)

Mass spectrometry (EI-MS) m/z: 586 (M⁺), 542, 415, 332, 288, 287,

Example 14 ¹³C-FDO Breath Test

As in 12-1, a ¹³C-FDO breath test was carried out wherein ¹³C-FDOdissolved in olive oil was orally administered (200 mg/kg) to chronicpancreatitis and control rats and the time course of the ¹³Cconcentration in the exhaled CO₂ after the administration was measured.

The Δ¹³C (%) values of the chronic pancreatitis rats were smaller thanthe control rats at each time point for 5 hours (FIG. 11). At 1 hourafter the administration, the Δ¹³C (%) value of 2.7±3.8 in the chronicpancreatitis rats was very significantly smaller than the value of46.3±11.5 in the control rats (p<0.001). At 3 hours after theadministration, the Δ¹³C (%) value of 18.4±12.1 in the chronicpancreatitis rats was very significantly smaller than the value of60.6±12.7 in the control rats (p<0.01). At 4 hours after theadministration, the Δ¹³C (%) value of 15.2±13.4 in the chronicpancreatitis rats was very significantly smaller than the value of88.7±8.2 in the control rats (p<0.001). At 5 hours after theadministration, the Δ¹³C (%) value of 20.6±11.2 in the chronicpancreatitis rats was very significantly smaller than the value of73.6±14.1 in the control rats (p<0.01).

Example 15 Preparation of ¹³C-diacetylfluorescein (¹³C-FDA)

Two grams (2 g) of 1- ¹³C-acetic acid (Masstrace) and 3.63 g offluorescein 2Na were dissolved in DMF and 15.94 g of BOP and 17.1 ml ofDIEA were sequentially added and stirred at room temperature for 12hours. After the completion of the reaction was confirmed by TLC, thereaction mixture was extracted with ethyl acetate. The organic layer wasdried, concentrated, purified through a column (solvent: 20% ethylacetate/hexane) under dark, and concentrated to yield 1.01 g of ¹³C-FDA.

The structure and ¹³C-labeled position were confirmed by ¹³C-NMR andmass spectrometry.

¹³C-NMR (heavy chloroform, 400 MHz): 168.8 ppm (¹³ COOR)

Mass spectrometry (EI-MS) m/z: 418 (M⁺), 374, 331, 314, 288, 287.

Formulation Example 1 Liquid for Internal Use

Purified water was added to 1.5 parts by weight of ¹³C-labeledcyclodextrin to produce a total of 100 parts by weight and this totalwas dissolved and sterilized through a Millipore filter. The filtratewas placed into a vial bottle and sealed to yield a liquid for internaluse.

Formulation Example 2 Liquid for Internal Use

¹³C-labeled cyclodextrin was mixed with an equal amount of non-labeledcyclodextrin. Purified water was added to 3 parts by weight of themixture to produce a total of 100 parts by weight and this total wasdissolved and sterilized through a Millipore filter. The filtrate wasplaced into a vial bottle and sealed to yield a liquid for internal use.

Formulation Example 3 Liquid for Internal Use

Olive oil was added to 4 parts by weight of ¹³C-FDL to produce a totalof 100 parts by weight and this total was dissolved, placed into a vialbottle and sealed to yield a liquid for internal use.

Advantages of the Invention

The present invention provides a test for pancreatic exocrine functionwhich imparts a low stress on subjects and gives the results in a shortperiod of time.

In the test, a ¹³C- or ¹³C-labeled oligosaccharide or polysaccharide ora salt thereof or a derivative thereof, or a cyclodextrin inclusioncomplex or a salt, thereof, ¹³C- or ¹⁴C-labeled fluorescein estercompound or a salt thereof is used. Among these materials, cyclodextrinsand non-reducing terminal modified oligosaccharides or polysaccharidesare useful as substrates for evaluating α-amylase secretion abilitysince they are specific to α-amylase in the digestive tract and are notdegraded by α-glucosidase (maltase). These properties are different fromthose of starch. Further, inclusion complexes using cyclodextrin whichis a substrate specific for α-amylase in the digestive tract providesubstrates for more universally carrying out a test specific to thedisease conditions by selecting the molecules included therein. Thesemethods impart much lower stress on subjects and require less skills foroperators compared to the conventional intubation test.

This test method may be utilized in diagnosis for pancreatitis in acollective physical examination, assessment of the seriousness ofchronic pancreatitis, precognition of onset of serious fulminantpancreatitis with a still high mortality (30%), diagnosis of causes forpancreatitis, and early diagnosis of pancreatic cancer. Further, it maybe useful as a diagnostic method for ruling out pancreatitis in medicalexamination of general outpatients.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entity.

What is claimed is:
 1. A ¹³C- or ¹⁴C-labeled fluorescein ester compoundor a salt thereof, resulting from a reaction of a ¹³C- or ¹⁴C-labeledacid with fluorescein at both or either of the two hydroxyl groups at 3′and 6′ positions to form an ester linkage.
 2. The compound or saltthereof according to claim 1, wherein the acid is a carboxylic acid. 3.The compound or salt thereof according to claim 2, wherein thecarboxylic acid is a fatty acid.
 4. The compound or salt thereofaccording to claim 3, wherein the fatty acid has 2 to 16 carbon atoms.5. The compound or salt thereof according to claim 4, wherein the fattyacid is selected from the group consisting of lauric, acetic andoctanoic acids.
 6. A ¹³C-labeled fluorescein ester compound or a saltthereof selected from the group consisting of the following compounds:a. ¹³C-dilaurylfluorescein; b. ¹³C-diacetylfluorescein; and c.¹³C-dioctanoylfluorescein.
 7. A method for diagnosing pancreaticexocrine function, comprising administering ¹³C- or ¹⁴C-labeledfluorescein ester compound or a pharmaceutically acceptable salt thereofto a subject.
 8. The method according to claim 7, comprising a compoundor salt thereof resulting from a reaction of a ¹³C- or ¹⁴C-labeled acidwith fluorescein at both or either of the two hydroxyl groups at 3′ and6′ positions to form an ester linkage.
 9. The method according to claim8, wherein the acid is a carboxylic acid.
 10. The method according toclaim 9, wherein the carboxylic acid is a fatty acid.
 11. The methodaccording to claim 10, wherein the fatty acid has 2 to 16 carbon atoms.12. The method according to claim 7, wherein the ¹³C-labeled fluoresceinester compound or the pharmaceutically acceptable salt thereof isselected from the group consisting of the following compounds: (a)¹³C-dilaurylfluorescein; (b) ¹³C-diacetylfluorescein; and (c)¹³C-dioctanoylfluorescein.
 13. The method according to claim 7, whereinthe ¹³C- or ¹⁴C-labeled fluorescein ester compound or pharmaceuticallyacceptable salt thereof is subjected to the reaction of the pancreaticexocrine cholesterol ester hydrolase and pancreatic exocrine esteraseand decarboxylated to generate ¹³CO₂ or ¹⁴CO₂.